DESCRIPTION (Applicant's abstract): The broad, long-term objectives of this application are to understand the mechanism by which mutations in the human cationic trypsinogen gene (PRSS1) lead to hereditary pancreatitis (HP). HP is an autosomal dominant genetic disorder characterized by early-onset recurrent attacks of acute pancreatitis with frequent progression to chronic pancreatitis and occasionally to pancreatic cancer. HP belongs to the inherited forms of idiopathic chronic pancreatitis, a genetically heterogeneous disease group, where mutations have been found not only in PRSS1, but also in the cystic fibrosis transmembrane conductance regulator (CFTR) gene and in the pancreatic secretory trypsin inhibitor gene (SPINK1). HP has been widely recognized as a highly relevant model system for all forms of human pancreatitis. In the majority of cases, three mutations, Arg117-His, Asn2l-,lle, and Ala8-Val, have been identified in PRSSI. The molecular defects caused by the HP mutations will be studied within the context of a current pathogenesis model, which suggests that HP is caused by excessive intrapancreatic trypsin activity via one of 3 mechanisms: (i) increased trypsinogen activation, (ii) decreased trypsin degradation; or (iii) impaired inhibition by pancreatic secretory trypsin inhibitor (PSTI). The principal objective of the experimental design is to study the effects of the HP-mutations in vitro, using recombinant human trypsinogens. Wild-type and mutant trypsinogens will be expressed in Escherichia coli, and purified to homogeneity with ecotin affinity chromatography. Catalytic properties and autocatalytic degradation (autolysis) of trypsins and autoactivation and autocatalytic degradation (zymogenolysis) of trypsinogens will be characterized. In addition, interactions between wild-type and mutant forms of cationic trypsin(ogen) with anionic trypsin(ogen) and mesotrypsin(ogen) will be examined. Finally, inhibition of human trypsin isoforms and HP mutant trypsins by wild-type and mutant PSTI proteins will be studied.